Bacterial luciferase reporters: the Swiss army knife of molecular biology

Bioluminescence is a process during which light in the visible spectrum is emitted as a consequence of an enzymatic reaction catalyzed by luciferases. Luciferases have been identified mainly in marine organisms and are used for several biological purposes include camouflage, repulsion, attraction, communication and illumination. Some of the currently known luciferases have become indispensible tools in modern molecular biology and are used for diverse applications such as autoinducer-1 activity assays, promoter test assays in both prokaryotes and eukaryotes, imaging of bacterial infections in live animals, in vivo activity assays genes involved in host response and disease and monitoring of bacterial contaminations of food products. With the present review, the authors intend to give an overview on the currently used bacterial luciferase reporter systems, their methodologies and applications and compare them to other reporter systems.     

Freshwater biogeography and limnological evolution of the Tibetan Plateau--insights from a plateau-wide distributed gastropod taxon (Radix spp.)

Background

The Tibetan Plateau is not only the highest and largest plateau on earth; it is also home to numerous freshwater lakes potentially harbouring endemic faunal elements. As it remains largely unknown whether these lakes have continuously existed during the Last Glacial Maximum (LGM), questions arise as to whether taxa have been able to exist on the plateau since before the latest Pleistocene, from where and how often the plateau was colonized, and by which mechanisms organisms conquered remote high altitude lentic freshwater systems. In this study, species of the plateau-wide distributed freshwater gastropod genus Radix are used to answer these biogeographical questions.

Methodology/Principal Findings

Based on a broad spatial sampling of Radix spp. on the Tibetan Plateau, and phylogenetic analyses of mtDNA sequence data, three probably endemic and one widespread major Radix clade could be identified on the plateau. Two of the endemic clades show a remarkably high genetic diversity, indicating a relatively great phylogenetic age. Phylogeographical analyses of individuals belonging to the most widely distributed clade indicate that intra-plateau distribution cannot be explained by drainage-related dispersal alone.

Conclusions/Significance

Our study reveals that Radix spp. persisted throughout the LGM on the Tibetan Plateau. Therefore, we assume the continuous existence of suitable water bodies during that time. The extant Radix diversity on the plateau might have been caused by multiple colonization events combined with a relatively long intra-plateau evolution. At least one colonization event has a Palaearctic origin. In contrast to freshwater fishes, passive dispersal, probably by water birds, might be an important mechanism for conquering remote areas on the plateau. Patterns found in Radix spp. are shared with some terrestrial plateau taxa, indicating that Radix may be a suitable model taxon for inferring general patterns of biotic origin, dispersal and survival on the Tibetan Plateau.     

Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells

The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.     

Current status of the E23K Kir6.2 polymorphism: implications for type-2 diabetes

The ATP-sensitive potassium (K_ATP) channel couples membrane excitability to cellular metabolism and is a critical mediator in the process of glucose-stimulated insulin secretion. Increasing numbers of K_ATP channel polymorphisms are being described and linked to altered insulin secretion indicating that genes encoding this ion channel could be susceptibility markers for type-2 diabetes. Genetic variation of K_ATP channels may result in altered -cell electrical activity, glucose homeostasis, and increased susceptibility to type-2 diabetes. Of particular interest is the Kir6.2 E23K polymorphism, which is linked to increased susceptibility to type-2 diabetes in Caucasian populations and may also be associated with weight gain and obesity, both of which are major diabetes risk factors. This association highlights the potential contribution of both genetic and environmental factors to the development and progression of type-2 diabetes. In addition, the common occurrence of the E23K polymorphism in Caucasian populations may have conferred an evolutionary advantage to our ancestors. This review will summarize the current status of the association of K_ATP channel polymorphisms with type-2 diabetes, focusing on the possible mechanisms by which these polymorphisms alter glucose homeostasis and offering insights into possible evolutionary pressures that may have contributed to the high prevalence of K_ATP channel polymorphisms in the Caucasian population.This work was supported by funding from the Canadian Diabetes Association (CDA) in honor of Gordon M. Stevenson, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Canadian Institutes of Health Research (CIHR). M.J.R. is supported by AHFMR and CDA Scholarships. P.E.L. received salary support as an AHFMR Scholar and CIHR New Investigator     

Edge effects on vegetation and soils in a Virginia old-field

We investigated the effect of proximity to forest edge on plant community structure and ecosystem properties during succession, using field measurements of leaf area index (LAI), species composition, and soil carbon. Data were collected along four transects within a 14-year-old temperate successional field in north-central Virginia over the 2000 growing season. Additionally, the normalized difference vegetation index (NDVI) was calculated from LANDSAT 7 satellite data at a resolution of 30 m for the entire field. Results showed that relative frequencies of trees increased with proximity to forest edge suggesting a more advanced stage of succession in areas close to the adjacent secondary forest. Significant negative relationships were observed between distance from forest edge and both peak season LAI and NDVI. LAI and NDVI values within 60 m of the forest edge, however, were not significantly different from those values in the adjacent mature secondary forest, suggesting that some community level properties may take relatively short periods of time to reach undisturbed states. The presence of several key plant species, particularly Celastrus scandens (climbing bittersweet), exhibited a strong control on the spatial variability of LAI and potentially the aboveground net primary production. Soil carbon levels did not show a significant increase at sites close to the adjacent secondary forest (relative to an adjacent crop field), as seen with LAI and NDVI, suggesting no recovery of soil carbon in these systems after 14 years. This study points to the complexity of factors that influence spatial patterns of succession in old-fields and suggests that invasive species may play an important role in successional pathways and carbon cycling.     

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture

Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation.     

Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

Role of afferent pathways of heat and cold in body temperature regulation

The detection of surface and internal temperatures is achieved by axons terminating at lamina I of the spinal dorsal horn, otherwise approached only by nociceptive afferents. Recent advances in thermal physiology research have disclosed that temperature-sensitive ion channels belonging to the transient receptor potential family exist in the peripheral sensory neurons and in the brain. Thermosensory, nociceptive and polymodal afferents project to different thalamic nuclei, and specific pathways to the insular cortex evoke the conscious experience of thermal sensation. The posterior insular region represents discriminative thermal sensation, while the largest correlation with subjective ratings of temperature is located in the orbitofrontal and anterior insular cortex. The insular cortex forms an integrative part of the limbic system and is closely tied with the hypothalamus, the amygdala, the anterior cingulate cortex and the orbitofrontal cortex and emerges as the main coordinator of behavioral, autonomic and endocrine responses to both non-noxious and noxious thermal stimuli. The firing rate of warm and cold receptors is not altered by pyrogens. A strong correlation between the onset of fever and production of superoxide by macrophages following the injection of pyrogens implicates reactive oxygen species as elicitors of fever, a hypothesis strengthened by the observation that oxygen radical scavengers or thiol reductants act as antipyretics. Oxidative stress appears to be sensed by the brain and a likely structure for its detection may be the redox-sensitive site of the N-methyl-d-aspartate (NMDA) receptor for glutamate, in that oxidation of this site causes fever while its reduction lowers body temperature, effects which are abrogated by specific NMDA receptor blockers.